e-Research: A Journal of Undergraduate Work - Chapman University

A. Cyr, M. Weitzman
Western Blotting
10-well and 12-well 4-12% Bis-Tris Acetate gels with MOPS running buffer to run 18 ul samples of 30 ug samples
and 60 ug samples respectively and full range rainbow recombinant protein marker. 1:5 loading dye prepared with
LDS sample buffer (4X) and IM DTT (20X). Gels ran at 150 V for 1.5 hours. Transfers ran at 30 V for 2 hours in 20X
NUPAGE transfer buffer, methanol, H2O, NUPAGE antioxidant. Membranes soaked in 5% milk with 100 ul NaN3
after transfer in 4 degree C cold room. Membranes washed with 1X PBST. Membranes soaked with mouse ICP0,
mouse GAPDH, or mouse Flag primary antibodies then horse-radish peroxidase-conjugated mouse secondary
antibodies in 5% milk. Equal volumes of Western Lightening Plus-ECL oxidizing reagent and enhanced luminal
reagent distributed over membranes for film development.
RESULTS
Cloning
Cloning the two separate constructs of RNF168 was carried out as described in the methods section. Correct
constructs of the clones were identified by restriction digest analysis and computer-based DNA sequencing. The
verified clones of F1 and F2 were purified and used in transfection experiments in the HeLa cells. As seen in figure
2, the F1 construct (~850bp) contained the RING finger motif and MIU1, and the F2 construct (~1Kbp) contained
MIU2.
Figure 2: Schematic images of F1 and F2 constructs of RNF168 cloned and transfected +/- ICP0.
F2 is degraded by ICP0 expression
ICP0 expression degrades several cellular substrates, but this study focused on RNF168 degradation specifically.
Degradations of the fragments from the separated RNF168 were compared to each other and to the full-length
RNF168. The lysates of ICP0 co-transfections in HeLa cells were analyzed by a western blot for F2. F2 fragment
degradation is evident with ICP0 expression as seen in Figure 3. The F2 fragment is not degraded with pcDNA3.1
vector alone in the absence of ICP0. F2 is sized at approximately 50kDa.
F1 may be degraded by ICP0 expression less efficiently
The western blot in Figure 3 indicates moderate degradation of F1 by ICP0 expression. Two separate lysates of F1
(F1-12 and F1-15) were analyzed on the western blot to express F1 in duplicate due to uncertainty of the quality of
the sequences of the clones. F1 degradation appears to be less efficient in comparison to F2 degradation. Both F1-
12 and F1-15 fragments suggest no degradation in the presence of pcDNA3.1 vector alone. F1 is sized at
approximately 38kDa.
42 e-Research, Vol 2, No 2 (2011)